粒線體基因組 (mitochondrial DNA; mtDNA) 編碼13種參與在電子傳遞鏈中的蛋白質，對於生物體功能是不可或缺的。然而相比起細胞核，粒線體卻更容易遭受氧化傷害而導致mtDNA受損。為因應此傷害，鹼基切除修復或是單股DNA斷裂修復都是粒線體內有效率的DNA修復機制。然而，如果基因受損的程度超過修復系統的負荷，將導致受損mtDNA的不正常累積，最終導致疾病。為即時清除受損的mtDNA，粒線體具有降解雙股DNA的機制。有趣的是，此降解過程是由粒線體內負責複製mtDNA的DNA聚合酶? (DNA polymerase ? ; POL?) 所執行。此酵素透過其三端至五端外切酶的活性，也就是DNA聚合酶的校對活性，可以降解受損的mtDNA片段。從目前已解開的POL?分子結構，DNA受質均結合在聚合酶的活性中心，距離此酵素的外切酶活性中心有約35 Å的距離。藉此觀察，我們推測當POL?在降解雙股DNA片段或是執行其校對功能時，其分子構型將與目前已知的結構有很大的不同。為揭示POL?在降解mtDNA，與執行校對功能時DNA受質穿梭於兩活性中心的結構機制，在此，我們將透過X-ray晶體學的方式，嘗試解析DNA受質結合於人類POL?外切酶活性中心的分子結構。目前，我們已成功純化出POL?的催化亞基，POL?A。並透過酵素活性及受質結合測試的結果，我們設計出一個含有髮夾結構以及在尾端帶有兩個核苷酸長度的叉形DNA，作為與POL?共結晶的受質。結晶條件的測試正在進行中。我們預期此研究能夠闡明POL?參與mtDNA降解的結構機制，並拓展我們對於mtDNA品質控管之分子機制的了解，以期對相關疾病的致病機轉與治療方針的發展，有所助益。

By encoding 13 proteins for electron transfer chain, mitochondrial DNA (mtDNA) is indispensable for mitochondrial function. However, comparing to nucleus genome, mtDNA faces more insults from reactive oxygen species that are constantly generated inside the organelle. To deal with the problem, mitochondria possess efficient base excision repair and single-stranded repair machineries. In addition, if damages overwhelm the repair system, a DNA degradation mechanism is triggered to actively eliminate damaged mtDNA copies. The timely clearance of damaged mtDNA, especially those are fragmented by double-stranded breaks, is crucial for preventing deleterious rearrangement of the genome. Intriguingly, this degradation process was found to be dependent on DNA polymerase ? (POL?), the sole replicase in mitochondria. In which the polymerase digests fragmented mtDNA through its 3ʹ-to-5ʹ exonuclease (i.e., proofreading) activity. In the available structures of POL? holoenzyme bound with a primed DNA template, the catalytic center of exonuclease domain is separated from polymerase core far away by approximately 35Å. With this notion, one can assume a large conformational change to accommodate substrate binding in exonuclease domain must occur during the enzyme digesting DNA or performing proofreading. Here we set out to reveal the molecular structure of POL? in performing 3ʹ-to-5ʹ exonuclease activity by X-ray crystallography. To this aim, we have successfully purified POL?A (the catalytic subunit of POL?) to homogenous. By activity and substrate-binding assays, we found that POL?A can only digest single-stranded DNA region but require duplex region for optimal substrate alignment. Accordingly, we designed a fork-shaped DNA consisting of a hairpin and 2-nt fork as the substrate for co-crystallizing with POL?A. Crystallization screen over available commercial crystallization kits in progress. We anticipate our work will uncover the structural basis of POL in degrading mtDNA and further our understanding of the molecular mechanism of mtDNA maintenance.

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